Purification and characterization of Atlantic salmon growth hormone and evidence for charge heterogeneity.
Highly purified growth hormone (GH) has been isolated from Atlantic salmon (Salmo salar) pituitaries by extraction with acid acetone, acidic precipitation, and reversed-phase high-performance liquid chromatography (HPLC). The yield was 2.5 mg/g wet tissue. The Atlantic salmon GH (sGH) emerged as a single symmetrical peak after HPLC on a reverse phase C18 column. SDS-gel electrophoresis revealed only one band with an estimated molecular weight of 23,000. Atlantic sGH showed a uniform molecular weight, but two-dimensional (2D) gel electrophoresis of the purified sGH revealed charge heterogeneity with pI's ranging from 6.5 to 8.2. Treatment of the purified sGH with alkaline phosphatase concentrated these different forms into a single more alkaline position (pI 8.2) indicating removal of acidic groups. These results were documented using both silver- and immunostaining of the 2D SDS gels. The purified sGH was phosphorylated in vitro by a calmodulin-dependent protein kinase. Phosphorylation of sGH may be a post-translational modification resulting in several molecular forms with variable acidity. Analysis of the amino acid composition of Atlantic sGH revealed homology with GHs isolated from other teleost species and the amino-terminal sequence showed only three different amino acids within the first 25 residues compared to GH isolated from chum salmon (Oncorhynchus keta) and coho salmon (Oncorhynchus kisutch) pituitaries. Atlantic sGH had a methionine as the amino-terminal residue. Antibodies against chum sGH cross-reacted with Atlantic sGH. Antibodies against either Atlantic or chinook (Oncorhynchus tschawytscha) salmon prolactin or human GH did not cross-react with Atlantic sGH. Atlantic sGH was shown to have a slight growth-promoting activity in the rat tibia assay.
Skibeli
V
V
Institute of Medical Biochemistry, University of Olso, Norway.
Andersen
O
O
Gautvik
K M
KM
D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001267:Atlantic Ocean; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D013006:Growth Hormone / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D015151:Immunoblotting; D007526:Isoelectric Point; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D010902:Pituitary Gland / Q000737:chemistry; D051381:Rats; D012474:Salmon / Q000378:metabolism*
D000596:Amino Acids; D013006:Growth Hormone; D000469:Alkaline Phosphatase